Unlike B and T cells, NK cells do not express antigen-specific receptors, yet they can eliminate virus-infected cells and cancer cells without harming normal cells. An important component that provides specificity in target cell recognition is inhibition of NK cells by inhibitory receptors that recognize surface molecules called major histocompatibility complex (MHC) class I. MHC-specific recognition by inhibitory receptors on NK cells prevents killing of normal healthy cells. The major goal of this project is to elucidate the mechanism by which inhibitory receptors regulate NK cell activation. NK cells possess several sets of activating and inhibitory receptors that control different steps in cytotoxic lymphocyte-mediated killing of target cells, including conjugation of NK cells to target cells, polarization of lytic granules towards target cells, and degranulation. Prior engagement of NK cells with MHC class I molecules by inhibitory receptors allows for greater intrinsic responsiveness to subsequent activation stimuli through a process called NK cell licensing (aka education). The number of inhibitory receptors engaged with MHC and the strength of MHC binding to inhibitory receptors calibrate the potential responsiveness of each NK cell for cytotoxicity and cytokine secretion. It is still not clear how licensing affects different steps in NK cell cytotoxicity, such as the contribution of the beta2 integrin LFA-1, which is essential for tight conjugation with target cells and for lytic granule polarization. Binding of cytotoxic lymphocytes to the adhesion ligand ICAM-1 requires an open conformation of LFA-1, which is regulated by inside-out signals from other receptors. We have evaluated whether unlicensed NK cells have a defect in inside-out signaling to LFA-1 for conjugate formation or in outside-in signaling by LFA-1 for lytic granule polarization. Unlicensed NK cells did not form as many stable conjugates with target cells. The reduction of NK cell conjugation to target cells was not attributed to altered beta2 integrin LFA-1 properties but was instead due to reduced inside-out signaling to LFA-1 by activating receptors. For those unlicensed NK cells that did form conjugates, LFA-1-dependent granule polarization was similar to that in licensed NK cells. Thus, licensing through inhibitory receptors controls signals as proximal as inside-out signaling by activating receptors but not integrin outside-in signaling for granule polarization.